1. Field of the Invention
The present invention relates generally to methods for detecting the presence of or risk of developing cancer and more specifically to methods for detecting the presence of hypomethylation of the H19 gene and the IGF2 gene.
2. Background Information
The single greatest impediment to cancer diagnosis is the general requirement that the tumor itself must be detected directly. Efforts to identify genetic abnormalities in normal tissues of patients with cancer or at risk of cancer have been disappointing. For example, BRCA1 mutations are present in only about 1% of breast cancers. A small fraction of patients with colorectal cancer have predisposing mutations in the APC gene (>1%), causing adenomatous polyposis coli. An even smaller fraction show mutations in genes responsible for replication error repair (>2% of colon cancer patients, or much less than 1% of the population), show mutations in genes responsible for nucleotide mismatch error repair causing hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome).
Diagnostic methods such as microsatellite instability, require for identification that a patient already have a tumor. For example, microsatellite instability compares microsatellite marker length between the monoclonal tumor cell population and normal tissue derived from the same patient.
Family history still remains the most reliable diagnostic procedure for identifying patients at risk of cancer. A molecular diagnostic approach that might identify patients with cancer or at risk of cancer, using only normal tissue, would offer a decisive advantage for intervention and treatment.
Except for rare hereditary cancer syndromes, the impact of molecular genetics on cancer risk assessment and prevention has been minimal. Cancer surveillance has been effective for some cancers in which risk can be identified, for example colorectal cancer in familial adenomatous polyposis coli and hereditary nonpolyposis colorectal cancer (Markey, L., et al., Curr. Gastroenterol. Rep. 4, 404-413 (2002)), but these syndromes cumulatively account for less than 1% of cancer patients (Samowitz, W. S., et al., Gastroenterology 121, 830-838 (2001); Percesepe, A., et al., J. Clin. Oncol. 19, 3944-3950 (2001)). Nevertheless, genetics is thought to contribute substantially to cancer risk, since the odds ratio for malignancy increases in patients with first degree relatives with cancer, e.g., 2 to 3-fold in colorectal cancer (Fuchs, C. S., et al., N. Engl. J. Med. 331, 1669-1674 (1994)). Therefore, there remains a need to develop genetic tests to identify these patients.
Accordingly, no tests are available for identifying common cancer risk in the general population. As discussed above, genetic abnormalities that are known to predispose to cancer are rare. At the same time, advances in cancer treatment have had a small impact on morbidity and mortality. A major advance in cancer requires identification of patients at risk (i.e. identifies patients before they develop cancer), which could be combined with increased surveillance and chemoprevention, similar to the modern approach to cardiovascular medicine.
Thus, there remains a need for a diagnostic method for detecting and/or screening for the presence of diseases and/or the risk of developing a disease. In particular, there remains a need for a method for detecting and/or screening for the presence of cancer, for example colorectal cancer. There also remains a need for a method of detecting and/or screening for the presence of cancer and/or the risk of developing cancer that can be applied to a wide section of the population.
Epigenetic alterations in human cancer, i.e., alterations in the genome other than the DNA sequence itself, were first described in 1983 by Feinberg and Vogelstein (A. P. Feinberg et al., Nature (Lond.), 301: 89-92, 1983), who found widespread hypomethylation of genes in CRCs and in premalignant adenomas. Epigenetic abnormalities identified subsequently include global genomic hypomethylation (A. P. Feinberg et al., Cancer Res., 48: 1159-1161, 1988), promoter hypermethylation of CpG islands (S. B. Baylin, et al., Cancer Res., 46: 2917-2922, 1986; A. Merlo et al, Nat. Med., 1: 686-692, 1995), and LOI (Rainier S. Johnson et al, Nature (Lond.), 362: 747-749, 1993; O. Ogawa et al., Nature (Lond.), 362: 749-751, 1993), or loss of the normal parent of origin-dependent gene silencing, affecting at least the genes IGF2, PEG1, p73, and LIT1 (S. Rainier et al., Nature (Lond.), 362: 747-749, 1993; O. Ogawa et al., Nature (Lond.), 362: 749-751, 1993; I. S. Pedersen et al, Cancer Res., 59: 5449-5451, 1999; M. P. Lee et al, Proc. Natl. Acad. Sci. USA, 96: 5203-5208, 1999; M. Kohda et al, Mol. Carcinog., 31: 184-191, 2001; Y. C., Cai et al., Carcinogenesis (Lond.), 21: 683-689, 2000; K. Tanaka et al, Oncology, 60: 268-273, 2001). LOI of IGF2 causes overexpression of IGF2 (J. D. Ravenel et al, J. Natl. Cancer Inst., 93: 1698-1703, 2001), an important autocrine growth factor in cancer. LOI was first identified in embryonal tumors in childhood, including Wilms' tumor, in which it is the most common molecular alteration (S. Rainier et al, Nature (Lond.), 362: 747-749, 1993; O. Ogawa et al, Nature (Lond.), 362: 749-751, 1993), as well as rhabdomyosarcoma (S. Zhan et al, J. Clin. Investig., 94: 445-448, 1994) and hepatoblastoma (S. Rainier et al, Cancer Res., 55: 1836-1838, 1995). LOI was also later found in common adult malignancies including ovarian (H. T. Kim et al, Am. J. Med. Genet., 80: 391-395, 1998), colon (H. Cui et al, Nat. Med., 4: 1276-280, 1998), lung (M. Kondo et al, Oncogene, 10: 1193-1198, 1995), and bladder cancer (M. Elkin et al, FEBS Lett., 374: 57-61, 1995), as well as chronic myelogenous leukemia (G. S. Randhawa et al, Blood, 91: 3144-3147, 1998). In CRC, LOI is particularly important because it is found commonly in both the tumor and normal tissue of patients with CRC, at ˜3-fold higher frequency then in patients without colon tumors (H. Cui et al, Nat. Med., 4: 1276-280, 1998), and, thus, LOI may represent the only common alteration linked to cancer that is found in normal tissue.
In Wilms' tumors, approximately half of tumors appear to arise by an epigenetic mechanism involving LOI rather than genetic alterations involving, for example, WT1 mutations and LOH, and the tumors with LOI appear in children who develop cancer at a later age, accounting for the bimodal age distribution of Wilms' tumor (J. D. Ravenel et al, J. Natl. Cancer Inst., 93: 1698-1703, 2001). LOI was linked to increased methylation, because Wilms' tumors with LOI of IGF2, i.e., activation of the normally silent maternal allele, show aberrant methylation of the normally unmethylated maternal allele of a DMR upstream of the H19 gene on the same chromosome (M. J. Steenman et al, Nat. Genet., 7: 433-439, 1994; T. Moulton et al, Nat. Genet., 7: 440-447, 1994). This result is consistent with the enhancer competition model for regulation of H19 imprinting. By this model, IGF2 and H19 promoters compete on the same chromosome for a shared enhancer, and access of the maternal IGF2 allele to this enhancer is blocked by the H19 DMR when unmethylated, likely because of the insulator activity of CTCF binding to the unmethylated H19 DMR (P. A. Leighton et al, Nature (Lond.), 375: 34-39, 1995; R. Ohlsson et al, Trends Genet., 17: 520-527, 2001; W. Reik et al, Nature (Lond.), 405: 408-409, 2000; A. T. Hark et al, Nature (Lond.), 405: 486-489, 2000; A. C. Bell et al, Nature (Lond.), 405: 482-485, 2000). Consistent with this, it has been observed that in Wilms' tumor, methylation of the maternal H19 DMR includes CTCF-binding sites (H. Cui et al, Cancer Res., 61: 4947-4950, 2001). These results would suggest that increased or ectopic activity of a DNA methyltransferase might lead to aberrant methylation of the maternal H19 DMR.
Therefore, it was surprising to observe that HCT116, a CRC line with normal imprinting of IGF2, is hypermethylated at H19 and retains normal imprinting after somatic cell knockout of the maintenance DNA methyltransferase DNMT1 but loses imprinting after subsequent somatic cell knockout of DNMT3B (Nature (Lond.), 416: 552-556, 2002), a de novo methyltransferase, i.e., that is able to methylate unmethylated sequences and is necessary for normal imprinting (M. Okano et al, Cell, 99: 247-257, 1999; K. Hata et al, Development (Camb.), 129:1983-1993, 2002). Therefore, there remains a need to determine the relationship between methylation state of IGF2 and H19 and loss of imprinting and/or cancer risk, such as colorectal cancer risk, and to devise methods for identifying cancer risk based on this relationship
The results described herein differ from past studies, and suggest a model of IGF2 imprinting in at least the colon that differs from the conventional view of enhancer competition between IGF2 and H19.